US 7674588 B2
Methods and apparatus are described for detecting specific binding between first and second chemical entities. The first chemical entity in association with a first fluorophore is immobilized. The second chemical entity is allowed to bind with the immobilized first chemical entity. The second chemical entity is or becomes coupled to a second fluorophore, which forms a FRET pair with the first fluorophore. The bound chemical entities are exposed to radiation at an excitation frequency for either the first or the second fluorophore, and polarization anisotropy of a FRET fluorescent signal from the bound chemical entities is measured to detect specific binding between the first and second chemical entities. Techniques are also disclosed for detecting whether a FRET interaction is occurring between a first chemical entity including a donor fluorophore and a second chemical entity including an acceptor fluorophore, using simultaneous anisotropy measurements at the wavelengths of the donor and acceptor fluorophores.
1. A method for detecting specific binding between a first chemical entity and a second chemical entity, the method comprising:
immobilizing the first chemical entity in association with a first fluorophore;
allowing the second chemical entity to bind with the immobilized first chemical entity, wherein the second chemical entity is or becomes coupled to a second fluorophore, which forms a FRET pair with the first fluorophore;
exposing the bound chemical entities to radiation at an excitation frequency for either the first or the second fluorophore; and
measuring polarization anisotropy of a FRET fluorescent signal from the bound chemical entities to detect specific binding between the first and second chemical entities,
wherein the polarization anisotropy is measured at a first wavelength and a second wavelength.
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23. A method for detecting whether a FRET interaction is occurring between a first chemical entity in association with a donor fluorophore and a second chemical entity in association with an acceptor fluorophore, the method comprising:
exposing at least one of the fluorophores to radiation at an excitation wavelength for the donor fluorophore;
measuring a polarization anisotropy of a fluorescent signal at an emission wavelength for the donor fluorophore and at an emission wavelength for the acceptor fluorophore; and
comparing the measured anisotropies to determine whether a FRET interaction is occurring between the first and second chemical entities.
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35. A method for detecting whether a FRET interaction is occurring between a first chemical entity in association with a donor fluorophore and a second chemical entity in association with an acceptor fluorophore, the method comprising:
exposing a region containing the fluorophores to radiation at an excitation wavelength for the donor fluorophore;
measuring a polarization anisotropy of a fluorescent signal at an emission wavelength for the acceptor fluorophore;
measuring a polarization anisotropy at the wavelength of a reference signal in the regions containing the fluorophores; and
comparing the measured anisotropies to determine whether a FRET interaction is occurring between the first and second chemical entities.
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This application is a continuation of U.S. application Ser. No. 11/398,496, filed on Apr. 4, 2006 now abandoned, and entitled “SCREENING USING POLARIZATION ANISOTROPHY IN FRET EMISSIONS”, which claims benefit of U.S. Provisional Patent Application No. 60/668,428, filed Apr. 4, 2005, and entitled “IN VITRO SCREENING USING POLARIZATION ANISOTROPY IN FRET EMISSIONS,” and of U.S. Provisional Patent Application No. 60/678,842, filed May 6, 2005, and entitled “SIMULTANEOUS TWO-COLOR DIFFERENTIAL ANISOTROPY IN FRET ASSAYS.” All of these applications are hereby incorporated by reference herein for all purposes.
This invention relates to improved Fluorescence Resonant Energy Transfer (FRET) techniques for use in high speed miniaturized assays for solutions, as well as in object-based and cell-based fluorimetry assays.
FRET provides an indication of proximity between donor and acceptor fluorophores. When a donor is excited with incident radiation at a defined frequency, some of the energy that the donor would normally emit as fluorescence is transferred to the acceptor, when the acceptor is in sufficiently close proximity to the donor (typically, within about 50 Angstroms for most donor fluorophores). At least some of the energy transferred to the acceptor is emitted as radiation at the fluorescence frequency of the acceptor. FRET is further described in various sources, such as “FRET Imaging” (Jares-Erijman, E. A, and Jovin, T. M, Nature Biotechnology, 21(11), (2003), pg 1387-1395), which is incorporated herein by reference for all purposes.
Another important concept in the context of this invention is anisotropy. Anisotropy provides a measure of the degree to which radiation is non-randomly polarized; that is, the degree to which one polarization orientation predominates over its orthogonal polarization orientation. A highly anisotropic signal will be highly polarized (for example, purely linearly polarized). A highly isotropic signal approaches random polarization. In one conventional approach, anisotropy (r) is calculated using the following equation:
Traditionally, FRET analysis relies on detecting one or more of the following: (1) the presence of fluorescence at the emission frequency of the acceptor, (2) the ratio of acceptor to donor fluorescence intensities, and (3) the lifetime of the donor's fluorescent emission. Each of these techniques has attendant difficulties. For example, merely detecting the presence of fluorescence at the emission frequency of the acceptor typically is not sufficient because the acceptor will produce some natural fluorescence when exposed to the frequency used to excite the donor fluorophore. Furthermore, time-resolved FRET imaging and analysis requires more complex instrumentation than standard fluorescence imaging and analysis.
Polarization anisotropy has been proposed as a FRET detection technique. Fluorescence generated from a FRET acceptor fluorophores is depolarized from the FRET process, and generally has relatively lower anisotropy than the fluorescence generated directly from donor fluorophores. Thus, anisotropy can be used as a measure of FRET, and, consequently, the associated proximity of donor and acceptor fluorophores. The use of this technique in homo-FRET, or FRET between like fluorophores, has been described in “Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)” (Lidke, D. S., Nagy, P., Barisas, B. G., Heintzmann, R., Post, J. N., Lidke, K. A., Clayton, A. H. A., Arndt-Jovin, D. J. and Jovin, T. M., Biochem. Soc. Trans., 31(5) (2003), pg. 1020-1027), which is incorporated herein by reference for all purposes.
Fluorescence anisotropy can also be employed as a FRET detection strategy in living cells. As has been described in “High contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy” (Rizzo, M. A. & Piston, D. W., Biophys J, 88 L14-16,2005), the fluorescence anisotropy for mCerulean, a type of Cyan Fluorescent Protein (CFP), has a value of about 0.3 across its entire wavelength emission range. However, when the mCerulean is in a FRET pair with mVenus, a type of Yellow Fluorescent Protein (YFP), the anisotropy remains high (slightly above 0.3) when the fluorescence is emitted by the donor (that is, the mCerulean) and decreases to about 0.15 when the fluorescence is emitted by the acceptor (that is, the mVenus). Although difference in anisotropy is very consistent between different measurements, the difference is relatively small and has limited use in experimental situations, since there are typically many unknowns and calibration factors which may affect the change in anisotropy. Thus it would be desirable to have, inter alia, an improved and more reliable method of measuring anisotropy changes associated with the FRET process.
One aspect of the invention pertains to methods and apparatus for detecting specific binding between a first chemical entity and a second chemical entity. The first chemical entity in association with a first fluorophore may be immobilized. The second chemical entity is allowed to bind with the immobilized first chemical entity. The second chemical entity is or becomes coupled to a second fluorophore, which forms a FRET pair with the first fluorophore. The bound chemical entities are exposed to radiation at an excitation frequency for either the first or the second fluorophore, and polarization anisotropy of a FRET fluorescent signal from the bound chemical entities is measured to detect specific binding between the first and second chemical entities.
Advantageous implementations can include one or more of the following features. In some cases, information in addition to anisotropy can be employed to evaluate a sample. For example, the method may include measuring donor emission lifetime of at least one of the first or second fluorophores. In some cases, the method measures the relative or absolute intensities of one or both fluorophores. In certain embodiments, the first and second fluorophores are the same (homo-FRET).
Many different chemical entities are contemplated for use with this invention. These may be small molecules, complexes, biomolecules such as proteins and nucleic acids, assemblies of molecules, biological structures such as organelles, etc. In certain embodiments, one of the first and second chemical entities comprises a protein and the other of the first and second chemical entities comprises a nucleic acid. In some embodiments, the method comprises performing a defined treatment on at least one of the first and second chemical entities, such that specific binding between the first and second chemical entities only occurs when the defined treatment results in at least one of the first and second chemical entities entering a particular state (e.g., a conformational state, a chemical modification such as phosphorylation, etc.).
Note that depending on the assay design, the polarization anisotropy may be measured at a single wavelength or multiple wavelengths. More typically, the fluorophores will be different. In some embodiments, the anisotropies of first and second wavelengths are measured simultaneously. In some embodiments, the anisotropies of the first and second wavelengths are measured sequentially in a time frame that is short with respect to a systematic measured change in signal readout. An example of a systematic measured change is biomolecular transition in a cascade of reactions in a biochemical pathway. The assay is designed to measure the biomolecular transition.
Another aspect of the invention pertains to methods and apparatus for detecting whether a FRET interaction is occurring between a first chemical entity including a donor fluorophore and a second chemical entity including an acceptor fluorophore. The chemical entities are exposed to radiation at an excitation wavelength for the first fluorophore. A polarization anisotropy of a fluorescent signal from the chemical entities is measured at an emission wavelength for the donor fluorophore and at an emission wavelength for the acceptor fluorophore. The measured anisotropies are compared to determine whether a FRET interaction is occurring between the first and second chemical entities. The anisotropy of signal at the emission wavelength for the donor fluorophore may provide an internal reference for calibrating the anisotropy measured at emission wavelength for the acceptor fluorophore. In other embodiments, the anisotropy of the emission wavelength for the acceptor fluorophore is compared with an “external” reference such as the anisotropy of the excitation source radiation or a separate fluorophore, unconnected with the FRET pair, which is provided in the assay sample. In the latter example, the external reference may be provided by, for example, a bead or a spot of fluorophore provided within or proximate to the sample.
Advantageous implementations can include one or more of the following features. In certain embodiments, a FRET interaction is determined to occur when the measured anisotropy at the emission wavelength of the acceptor fluorophore changes relative to the measured anisotropy at the emission wavelength of the donor fluorophore. In some cases, the anisotropies of the donor fluorophore and acceptor fluorophore are measured simultaneously. In certain embodiments, the anisotropies of the first and second wavelengths are measured sequentially in a time frame that is short with respect to a systematic measured change in signal readout.
The first and second chemical entities may be included in a target region for FRET analysis. Examples of target regions include one or more of microbeads, spots, spot on spot, spot on a slide combined with a bead, samples or objects confined in a capillary tube, samples or objects confined in a microfluidic channel, aggregates or colonies of cells confined in a liquid region such as a well or droplet, and aggregates or colonies of cells immobilized on a surface or surface layer.
Many different types of interactions may be compared using methods of this invention. In one example, at least one of the chemical entities is a small molecule that becomes loses some conformation flexibility upon binding to the other chemical entity. Many different fluorophore combinations may be employed. In some cases, the donor fluorophore and the acceptor fluorophore are the same. The chemical entities may be associated with fluorophores in many different ways. In one example a conventional binding mechanism is employed such as an antibody-antigen interaction or a strepavidin-biotin interaction.
The invention can be implemented to include one or more of the following advantages. Using array-based assays in which polarization anisotropy in FRET is employed to detect specific binding does not require time-based signal capture and does not require collection at multiple wavelengths (that of the donor and acceptor fluorophore emissions), unless two-color anisotropy measurements are made on the array-based assays. It also provides a large dynamic range and high signal to noise ratio. FRET-based assays of this invention can be utilized in essentially any currently available assay format, including various types of sandwich assays. The assays may be employed to detect biological molecules as well as various non-biological molecules such as chemical warfare agents. The conventional assays are simply modified so that binding partners are each supplied with their own fluorophore, one being a donor fluorophore and the other being an acceptor fluorophore.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the description and drawings, and from the claims.
Like reference symbols in the various drawings indicate like elements.
In accordance with one aspect of the present invention, anisotropy is measured as part of a FRET detection strategy in assays, such as the mentioned array-based assays, to detect specific binding. In accordance with another aspect of the present invention, the invention provides methods and apparatus, implementing and using techniques for improved FRET detection and/or quantization, generally, as well as in the above-mentioned array-based assays.
The improved FRET detection techniques stem from the realization that a dye that functions as an acceptor in a FRET pair has significantly lower anisotropy than it has when it is not interacting in a FRET pair. A dye that functions as a donor in a FRET pair, however, has essentially the same anisotropy when it is the donor in a FRET pair and when it is not interacting as a donor in a FRET pair. Thus, by studying the ratio or difference between the anisotropy in the emission wavelength region of the acceptor and the emission wavelength region of the donor, it is possible to determine whether FRET interaction is occurring. Furthermore, the anisotropy of the donor's emission can work as an internal reference against which the anisotropy change of the acceptor's emission can be detected and/or quantified. In certain embodiments, apparatus in accordance with the invention allows simultaneous anisotropy measurements to be performed in two different wavelength regions. Thereby, both types of anisotropy are measured while all other parameters remain the same, which avoids any uncertainty that parameters might have changed, as would be the case if sequential measurements were performed. Furthermore, the apparatus allows the measurements and processing to be done in real time at the time of detection and subsequent digitization. This is referred to below as either “streaming data processing” or “on-the-fly” data processing.
Specific embodiments of the invention are described in detail below and illustrated in the accompanying drawings. While the invention will be described in conjunction with these specific embodiments, it will be understood that they are not intended to limit the invention to one embodiment. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention.
Many different types of instrumentation are suitable for analyzing anisotropy in arrays employing FRET systems to identify specific binding. A description of some suitable apparatus is provided in the following documents: U.S. patent application Ser. No. 10/927,748 (Published Application No. 2005-0046848-A1, published Mar. 3, 2005), filed Aug. 26, 2004, Entitled: Time Dependent Fluorescence Measurements, by inventors Cromwell et al., U.S. patent application Ser. No. 10/928,484 (Published Application No. 2005-0046849-A1, published Mar. 3, 2005), filed Aug. 26, 2004, Entitled: Measuring Time Dependent Fluorescence, by inventors Cromwell et al. and U.S. patent application Ser. No. 11/055,244, filed Feb. 9, 2005, Entitled: Methods And Apparatus For Scanning Small Sample Volumes, each of which is incorporated herein by reference for all purposes.
The resulting fluorescence from the sample is collected with two sets of collection optics (119) and passed to two respective detectors (121). The collection optics (119) can be configured to allow scanning of a large array, such as a microarray plate. In one embodiment, the collection optics (119) is a rod lens, designed to capture the entire range of sweep of the beam over one dimension of the base of the sample array. The collection optics (119) can also include other types of lenses, or an aggregate of lenses, as would be chosen for a particular assay type to capture the specific information required from the emission in such assay. In some embodiments, multiple setups of collection optics (119) can be used to improve collection efficiency.
The two sets of collection optics (119) allow simultaneous collection of separate components of emitted fluorescent light (e.g., separate wavelengths, polarization states, etc.). For example, a first polarizing filter can be used to pass only light of a first polarization to a first detector, and a second polarizing filter can be used to pass only light of a second, orthogonal, polarization to a second detector. The correlation of the signals collected in this configuration, detection in the detection system, and subsequent manipulation of the stored signal give rise to information not available to a single detector, with attendant improvement in signal. Thus, the information derived from this apparatus is steady-state anisotropy. Furthermore, when the apparatus is configured with appropriate detection circuitry to capture fluorescence lifetime data, it is possible to measure the correlation of time-dependent behavior of fluorescence anisotropy. As will be discussed in further detail below, this anisotropy measurement capability can be advantageously used to provide important information in FRET measurements.
The transmission of the fluorescent light to the detectors (121) can be accomplished by, for example, an optical fiber or a bundle of optical fibers (120). In one embodiment, the detectors (121) are detectors with high gain, such as photomultiplier tubes, which produce electrical output signals. It should be noted that any polarization filtering is typically performed before the collected light enters any optical fibers (120), since most optical fibers distort the polarization information and light that is output from an optical fiber does not have identical polarization components to the light that was input to the optical fiber at the other end. In the depicted embodiment, the output signals from the detectors are processed by ADC/DSP electronics (114) and passed on to a computer (124) for further processing. The computer (124) performs operations such as optimization of the gain and the signal to noise ratio (S/N), by making use of signal enhancing, averaging, or integrating detection systems.
In certain embodiments, the FRET detection apparatus can be very simple; it need only be outfitted to measure the polarization state of emitted radiation. However, more complex instrumentation may be employed to provide additional data relevant to FRET. For example, the instrumentation may also be capable of measuring the lifetime of fluorescent signals, particularly intensity as a function of time and/or anisotropy as a function of time. In a typical system, the detection circuitry is capable of recording and storing an emission signal (or multiple emission signals at different wavelengths) for a period of between about 0.5 nanoseconds and 10 nanoseconds after the excitation energy is removed from the sample. In some cases, depending upon the fluorophore employed, the measured duration of the emission signal may be in the hundreds of nanoseconds or even microseconds. Certain rare earth doped fluorophores have fluorescence lifetimes in the millisecond range. As indicated above, one manifestation of FRET is a reduced donor emission lifetime. The instrumentation may also have the ability to simultaneously capture emitted radiation at the frequencies of both the donor and the acceptor, or acceptor and some other reference radiation source with a known anisotropy. This allows the instrument to provide intensity ratio measurements as well. The above apparatus may be outfitted with redundant collectors for the different frequencies or it may employ a single collection system that alternately captures radiation at a reference frequency and a signal frequency.
As explained in more detail elsewhere herein, certain embodiments of the invention employ a reference signal to calibrate FRET signals, particularly the anisotropy, lifetime, and/or intensity of such signals. In many cases, the reference signal will be an internal reference taken from, for example, emission of a donor fluorophore in the FRET pair.
More generally, the internal reference for measurement may be provided from a molecular fluorophore having similar physical and/or chemical properties to the fluorophore in the sample whose signal is being measured (i.e., the acceptor fluorophore). This source of internal reference is often desirable because the physico/chemical interaction present in the reference fluorophore will presumably be acting in the same way as in the sample itself. Therefore, the analogous molecular internal reference provides an accurate internal calibrated reference polarization source. However, there may be circumstance where such reference signal may not be as advantageous to use. In such cases, an alternative reference source such as signal from the excitation source itself (e.g., a laser) is a suitable internal reference because it provides a particularly strong signal and is not dispersed among many wavelengths as the fluorescence signal normally is. Such reference signal can easily be measured in a scatter channel, and is not depolarized except by way of the measurement optics. In a similar way, for a spot array, a reference fluorophore can be spotted and made large and highly concentrated, so the fluorescence reference signal is very strong and easy to measure.
Another instance where an external reference may be desirable is the case where the internal reference chromophore undergoes homo-FRET, and this causes the internal anisotropy to become depolarized. In this case the internal reference may not be reliable, or may be systematically changing with conditions such as concentration, etc. So in this case an external reference may be more reliable, especially one that provides a strong signal that is present with the sample.
Anisotropy readouts of the FRET process may be advantageously employed in conventional array-based assays, such as ELISA, particularly those that suffer from high levels of non-specific binding. Such non-specific binding may occur at locations on a ligand other than the specific binding site of interest, producing background signal that interferes with analysis. For example, an analyte species sometimes attach to the Fc domain, rather than the variable domain, of an immobilized antibody, thereby providing a false indication of specific binding. By employing a FRET detection strategy, non-specific binding contributes relatively little to the collected signal and therefore does not significantly interfere with detection. Only those array locations where the donor and acceptor fluorophores are held in close proximity (typically the locations where specific binding has occurred) generate signal. As a result, FRET-based assays can provide improved signal-to-noise ratio over conventional array-based assays. However, the potential of FRET as an array-based detection technique has not been realized because of, for example, complicated instrumentation. Embodiments of the present invention provide array-based assays in which polarization anisotropy in FRET is used to detect specific binding.
In the examples presented in these figures, the protein-protein interaction detectable by FRET anisotropy takes place only when a residue (for example, tyrosine) is phosphorylated. Such phosphorylation may occur, for example, during signal transduction within a cell. Assays as depicted in the figures can provide information about the effects of various stimuli on the portion of the signal pathway involving tyrosine phosphorylation. For example, a particular treatment under investigation may possibly interfere with a kinase's ability to phosphorylate tyrosine in a protein involved in the signal pathway.
Of course, the invention is not limited to detecting interactomes (interaction features in a signal pathway) or any other class of reactions or molecular features. The assays of this invention can be applied to essentially any small dimension morphological or molecular feature. Features that can be assayed include protein domains, nucleic acid sequences, epitopes, various complexes, trascriptomes, and interactomes. The structure, physical and/or chemical features of essentially any chemical or biochemical species or moiety can be assayed. This includes protein-protein interactions, protein-nucleic acid interactions, and nucleic acid-nucleic acid interactions. Some examples of protein array assays are described in Zhu, Bilgin, and Snyder, “Proteomics”, Annu. Rev. Biochem. 16 (2003), pages 783-812, which is incorporated herein by reference for all purposes.
In a typical experiment, the transfected cells are exposed to a particular stimulus under investigation (for example, a pharmaceutical candidate) and allowed to grow. The cells are then lysed and the cellular contents are assayed, in some cases after being subjected to an initial separation procedure. In the example of
As was discussed above, certain aspects of the invention rely on the fact that a dye (fluorophore) with a high intrinsic anisotropy that functions as an acceptor in a FRET pair has significantly lower anisotropy than it has when it is not interacting in a FRET pair. A dye, also with a high intrinsic anisotropy, that functions as a donor in a FRET pair, however, has essentially the same anisotropy when it is a donor in a FRET pair and when it is not interacting as a donor in a FRET pair.
A fluorophore with a “high” intrinsic anisotropy may be understood to be a fluorescent species that interacts with linearly polarized excitation light by producing strongly polarized emission light. Quantitatively, the light emitted from a fluorophore with high intrinsic anisotropy will have a calculated anisotropy (using the expression in the Background section of this document) of at least about 0.3, more preferably at least about 0.4, when stimulated with light at the fluorophore's excitation frequency.
An inventive analysis method and system will now be described by way of example of an embodiment that uses the detection system as described above in conjunction with optically encoded beads and/or live cells. It should however be realized that the invention is not limited to beads and live cells, but any type of objects or combinations of objects suitable for FRET analysis can be used. Such examples of objects or combinations thereof include microbeads, spots, spot on spot, spot on a slide combined with a bead, samples or objects confined in capillary tubes or microfluidic channels, and so on. The central idea is to simultaneously measure anisotropy for two different wavelength regions for a sample confined in a well-defined target region, and to use one of the measured anisotropies as an internal reference for detecting and/or quantifying a change of the other measured anisotropy for the sample in the target region, thereby allowing for detection of the presence, absence and/or quantity of FRET in the target region. While the cases described herein use the donor fluorescence as a reference, other sources with known anisotropy can also be used as a reference, including other non-FRET fluorescent molecules present in the assay system or even scattered light signals from an excitation source. In another case, homo-FRET between molecules with high intrinsic anisotropy can be measured by observing the decrease in anisotropy upon FRET. In the case of homo-FRET, where the donor and acceptor have the same emission frequency, a separate reference emission with a known anisotropy and present in the sample can be used as an internal reference to improve quantitation. As the skilled person realizes, these techniques can be advantageously combined with the above-mentioned array based assays. In each case, a feature such as a homo-FRET pair, a scattering center, etc., that produces signal for the internal standard will be co-localized with the assay binding pair.
An experimental example will now be described with reference to
As can be seen in the two right-most bars, the FRET signal shows significantly increased separation of blue anisotropy and yellow anisotropy. In fact, the blue anisotropy has increased in comparison to the non-FRET example, while FRET-yellow anisotropy has decreased. The increase for the FRET-blue anisotropy likely stems from a decrease in fluorescence lifetime of the CFP because of FRET, resulting in slightly less opportunity for the blue donor fluorophore to undergo changes that would introduce randomness into the polarization state of its emitted frequency. In other words, the emission from the donor fluorophore is limited by transmission of some energy to the acceptor fluorophore that would otherwise go to emission of fluorescence radiation, and the remaining energy that is emitted by the donor has a high anisotropy. The FRET-yellow is a combination of the direct excitation of the CFP (some emission by CFP at the yellow end of spectrum), direct excitation of the YFP (by the radiation used to stimulate CFP), and a YFP emission resulting from FRET. The anisotropy will vary for each of these components, so what the rightmost bar represents is a weighted average of the anisotropy in each of these factors. The first two factors should result in relatively high anisotropy. Even so, the FRET-yellow average anisotropy is significantly lower than the other average anisotropies of
Next, an example will be presented showing how FRET can be identified for CFP/YFP transfected live cells.
A summary of certain process embodiments of the invention will now be provided.
The process depicted in
As shown in top-most block of
At some point in the process, the system obtains a measure of polarization anisotropy at a wavelength associated with an anisotropy reference. See block 1705. This may entail providing excitation radiation at a wavelength distinct from that applied in operation 1703. However, for many embodiments, a single excitation source—one at the excitation frequency of the donor—will be sufficient. As shown in block 1707, the process also involves obtaining a measure of polarization anisotropy at a wavelength at which the acceptor fluorophore emits radiation during FRET. With this information, the system may compare the measure of anisotropy at the emission wavelength of the acceptor with the measure of anisotropy at the wavelength of the anisotropy reference. See block 1709. Based on this anisotropy comparison, the process assesses the reaction or interaction between species A and species B. See block 1711.
This interaction or reaction mentioned in block 1701 is typically conducted in a designated sample region such as a small, sometimes confined area where species A, species B, the donor fluorophore and the acceptor fluorophore are mixed or otherwise allowed to come into intimate contact with one another. Typically, though not necessarily, the sample regions are wells or spot regions on a multi-well plate, or regions in a flow channel. Other examples include (a) in situ analysis of a tissue or organ sample, a blood flow, etc., (b) flat rotating substrates as described in U.S. patent application Ser. No. 11/055,244, filed Feb. 9, 2005 (incorporated herein by reference for all purposes), (c) in situ analysis of cells or compartments within cells such as the nucleus or cytoplasm, and (d) in situ analysis of aggregates of cells, such as cell colonies, where distinct regions or specific aggregates may be undergoing specific interactions causing a FRET signal.
As indicated above, the donor and acceptor fluorophores should be capable of forming a FRET pair. Many suitable fluorophore pairs are familiar to those of skilled in the art. In addition to the fluorescent protein pairs exemplified above, other fluorescent entities suitable for participating in FRET pairs include fluorescein, rhodamines, the Bodipy family of dyes (available from Invitrogen Corporation of Carlsbad, Calif.), ethidium bromide, fluorescent coumarins, cyanine dyes, etc. Various fluorescent proteins (typically provided as chimeric fusion proteins in vivo) may be employed. These include he red fluorescent protein cloned from Discosoma coral (DsRed or drFP583), green fluorescent protein (GFP), blue, cyan, and yellow variants of GFP, cyan fluorescent protein, and yellow fluorescent protein, to name a few.
Initially, when the components of the assay are brought together, species A and B may be either physically separated or linked. Separated species may be binding or reacting pairs that are brought into contact in order to assess whether the binding or reacting takes place in the assay sample. In such cases, the assay may begin with relatively high polarization anisotropy in the signal from the acceptor's emission frequency. But when or if the reaction takes place, there will be a detectable decrease in polarization anisotropy from the acceptor emission. In another case, when species A and B are initially linked and then exposed to conditions which might potentially cleave them or otherwise cause them to become further separated from one another, the reaction or interaction under investigation will result in a detectable increase in polarization anisotropy in signal collected at the acceptor's emission wavelength.
Regarding operation 1703, the excitation source for the assays described herein is typically linearly polarized or otherwise strongly polarized. It is provided in a narrow frequency band at the excitation frequency of the donor fluorophore, although this is not always necessary, particularly if the excitation frequency of the acceptor fluorophore, as well as the emission frequencies of the donor and acceptor fluorophores, is well removed from the excitation frequency of the acceptor. The intensity of the excitation radiation is chosen as appropriate for the assay under consideration. Typically, one wishes to provide a sufficiently high intensity to produce strong FRET signals, but not so high that it interferes with the species undergoing reaction. For many in vitro assays, the intensity range of the source (as it interacts with the sample—accounting for losses caused by the optics) will be between about 1×103 and 2×105 Watts/cm2. More typically, the range will between about 2×103 and 2×104 Watts/cm2.
Regarding operation 1705, a reference is provided for calibration or comparison of the polarization anisotropy signal from the sample. As mentioned, the local conditions of the assay other than FRET interactions may impact anisotropy independently. As a consequence, it is sometimes preferable to measure a relative, rather than an absolute value of anisotropy. Obtaining the polarization anisotropy from the reference may be accomplished as describe elsewhere herein, namely by determining the degree to which the reference signal is polarized. Typically this is. obtained by measuring signal at two orthogonal polarization angles, one parallel to the polarization direction provided by the excitation source and the other perpendicular to that direction. Significant differences in the intensity taken in the two directions indicates high anisotropy, whereas more equal values of intensity taken across the two directions indicates isotropy. Again, the anisotropy of the reference is taken for the purpose of calibrating the anisotropy measured for the sample's acceptor fluorophore emission.
As mentioned, a reference may be an internal reference or an external reference. An internal reference signal is typically obtained from the anisotropy of the donor fluorophore's emission signal. An external reference is taken from a source other than the donor's fluorescent emission. As mentioned, one example is emission from a reference, non-donor, non-acceptor fluorophore within the sample. Such reference may be immobilized on the sample containment vessel, provided on a bead within the sample, provided in solution, etc. Another example of an external reference is radiation from the excitation source as measured in the sample; for example scattered light collected from the sample.
Regarding operation 1707, the polarization anisotropy of the acceptor fluorophore's emission frequency is measured in the same way as the polarization anisotropy of the reference, but at a different wavelength. As explained above, depending on whether the interaction under investigation brings species A and B into closer proximity or separates them, a decrease or increase in polarization anisotropy at the acceptor's emission frequency will be a signature for the interaction.
Regarding operations 1709 and 1711, the comparison and assessment will typically take place using a computer, circuitry, or specially constructed logic. A threshold level of difference in anisotropy between the sample and the reference may be used to indicate that the reaction or interaction is taking place. Alternatively or in addition, a quantitative value of the difference in anisotropy may be used to indicate the extent to which the reaction or interaction is taking place. In certain embodiments, the relative anisotropy value provides an indication of the concentration or potency of species A or species B.
In some embodiments, prior to probing FRET interactions, the species and fluorophores are allowed to interact as necessary, sometimes for a defined period of time or after exposure to a particular stimulus such as elevated temperature, low oxygen, etc. See block 1805. After the sample under investigation is prepared, it is probed by exposing it to excitation radiation having a wavelength within the range fluorescence excitation wavelengths for the donor fluorophore. See block 1807.
After exposing the assay sample to excitation radiation, the system measures polarization anisotropy at a wavelength of fluorescence emission for the acceptor fluorophore. See block 1809. As explained above, this information is used to determine whether FRET is occurring. Based on the measured polarization anisotropy, the process assesses whether a putative reaction or interaction between species A and species B has taken place. See block 1811. If it is determined that the reaction took place, some embodiments of the invention optionally determine the degree to which it took place.
Certain embodiments of the invention pertain to a device, system or apparatus for performing the aforementioned operations. The system may be specially constructed for the required purposes, or it may be a general-purpose optical instrument, selectively activated or configured by, for example, a computer program stored in the computer. The processes presented above are not inherently related to any particular optical instrument or computing apparatus.
A number of implementations of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. For example, the above description has been focused on spot array and live cell formats for the assays. The invention is of course not limited in this manner. As another example, the solid substrate may comprise beads. In the spot array format, a wide variety of capture area sizes may be employed depending on the application. The spot density will also vary depending on the application. In a specific example, the substrate comprises wells and these sized as appropriate for the application; e.g., spot sizes of approximately 50 nl or smaller. The pattern of wells or spots may encode or provide particular information such as bar code information. In a specific application, the substrates are designed to provide patterns of antibody arrays for core members of signaling pathways. The pattern of spots may contain materials used to generate a reference measurement or control signal for either the assay or the signal readout, or may be simply used as a locating device (fiducial) for the assay spot array. Accordingly, other embodiments are within the scope of the following claims.